Decrease In mtDNA Copy Number Research Articles

Mitochondrial DNA depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human diffusely infiltrating astrocytomas

Mitochondrial DNA (mtDNA) alterations and their clinical and pathological implications have been analyzed in several human malignancies. A marked decrease in mtDNA copy number along with the increase in malignancy was observed in diffusely infiltrating astrocytomas (24 WHO grade II, 18 grade III, and 78 grade IV or GBM) compared to non-neoplastic brain tissues, being mostly depleted in GBM. Although high relative gene expression levels of mtDNA replication regulators (mitochondrial polymerase catalytic subunit ( POLG), transcription factors A ( TFAM), B1 ( TFB1M) and B2 ( TFB2M)) were detected, it cannot successfully revert the mtDNA depletion observed in our samples. On the other hand, a strong correlation among the expression levels of mitochondrial transcription factors corroborates with the TFAM role in the direct control of TFB1M and TFB2M during initiation of mtDNA replication. POLG expression was related to decreased mtDNA copy number, and its overexpression associated with TFAM expression levels also have an impact on long-term survival among GBM patients, interpreted as a potential predictive factor for better prognosis.

Depleción del ácido desoxirribonucleico mitocondrial y mutaciones de POLG en un paciente con neuropatía sensorial atáxica, disartria y oftalmoplejía

Background and objetive A broad spectrum of clinical disorders is produced by mutations in the DNA polymerase gamma mitochondrial ( POLG) gene which are associated with altered mitochondrial DNA (mtDNA) integrity. The majority of disorders characterized by multiple mtDNA deletions present with progressive external ophthalmoplegia, though this feature is not usually found in syndromes caused by mtDNA depletion. We report on a patient having the clinical triad of sensory ataxic neuropathy, dysarthria and ophthalmoplegia (SANDO), POLG mutations and reduced muscle mtDNA content. Patient and methods The patient presented with sensory ataxic neuropathy, dysarthria and ophthalmoplegia. Diagnosis was established by using histological and genetic procedures (nerve biopsy, mtDNA molecular analysis in skeletal muscle and mutation screening in the POLG gene). Results Sural nerve biopsy showed marked loss of large myelinated fibers. Skeletal muscle analysis revealed multiple mtDNA deletions, a marked decrease in mtDNA copy number and pathogenic mutations in the POLG gene. Conclusions POLG mutations must be considered in all patients with the cardinal findings of the SANDO phenotype, without taking into account the type of abnormalities encountered in the mitochondrial genome.

Discovery of a Novel Function for Human Rad51: MAINTENANCE OF THE MITOCHONDRIAL GENOME

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.

Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation

Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.

Mitochondrial DNA damage analysis in bronchoalveolar lavage cells of preterm infants

In mechanically ventilated preterm infants, the combination of immaturity, volutrauma, oxidative stress, and inflammatory processes can lead to chronic lung injury. Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA. We aimed to investigate the level of mtDNA damage (deletions, mutations and changes in copy number) in bronchoalveolar lavage (BAL) cells from 10 preterm infants (27-30 weeks). A first BAL (BAL1) was done within 24 h of endotracheal intubation and BAL2 was performed 30-103 h thereafter. Deletions were analyzed by long range PCR, point mutations by heteroduplex analysis of the D-loop region, and copy number changes by real-time PCR. Using these methods, no deletions were found in any of the BAL samples. When BAL1 and BAL2 samples were compared no new mutations were found. In contrast, a marked decrease in mtDNA copy number was observed in 5 patients. In conclusion, we found that exposure of preterm infants to short term mechanical ventilation did not lead to detrimental consequences for the mtDNA in the form of mutations or deletions.

Profound Length Heteroplasmic Alterations in Mitochondrial DNA Control Region From Primary AML Cells: Implication of Impaired Mitochondrial Replication and Demonstration of ‘vicious cycle' Hypothesis.

Abstract 3116Poster Board III-53Mitochondrial DNA (mtDNA) control region (displacement (D)-loop including HV1 and HV2) is a non-coding region of 1124 bp (nucleotide positions, np 16 024–576), which acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements (Blood 2004;103:4466-77). Importantly, mutations in the D-loop regulatory region might change mtDNA replication rate by modifying the binding affinity of significant trans-activating factors (Eur J Cancer 2004;40:2519-24). Thus, length heteroplasmic alterations of mtDNA control region may be related with mitochondrial dysfunction resulting in ‘vicious cycle' (Mol Med Today 2000;6:425-32). In an attempt to investigate profiling of mtDNA length heteroplasmic alterations in primary AML cells, we carried out a quantitative size-based PCR product separation by capillary electrophoresis (ABI 3130XL Genetic Analyzer and ABI Prism Genotyper version 3.1) using six targets (np 303-315 poly C, np 16184-16193 poly C, np 514-511 CA repeats, np 3566-3572 poly C, np 12385-12391 poly C and np 12418-12426 poly A). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research). Forty-eight AML bone marrow samples were collected after receiving Institutional Review Board approval and informed consent. There were profound alterations of mtGI in 303 poly C, 16184 poly C and 514 CA repeats. The length heteroplasmy pattern of 303 poly C tract in the HV2 region disclosed mixture of 7C, 8C, 9C and 10C mtDNA types. In the HV2 region, length heteroplasmy in poly-C tract at np 303 - 309 exhibited 5 variant peak patterns: 7CT6C+8CT6C (50.0%), 8CT6C+9CT6C (14.0%), 8CT6C+ 9CT6C+ 10CT6C (10.4%), 9CT6C+10CT6C+11CT6C (8.3%) 9CT6C + 10CT6C + 11CT6C+12CT6C (2.1%). The length heteroplasmy pattern of 514-523 CA repeats in the HV2 region exhibited 2 variant peak patterns: CACACACACA (56.3%) and CACACACA (43.7%). In the HV1 region, length heteroplasmy in the poly-C tract at np 16184 - 16193 exhibited 9 variant peak patterns: 5CT4C+5CT3C (31.0%), 6CT4C+6CT3C (2.1%), 9C+10C+11C+12C (16.7%), 9C+10C+11C (2.1%), T4CT4C+5CT3C (4.2%), 9C+10C+11C+12C+13C (2.1%), 3CTC4C+5CT3C (2.1%), 10C+11C+12C+13C (4.2%), 8C+9C+10+11C (2.1%). Primary AML cells revealed decreased enzyme activity in respiratory chain complex I, II and III. AML cells had about a two-fold decrease in mtDNA copy number compared with normal blood mononuclear cells. Current study demonstrates that profound length heteroplasmic alterations in mtDNA control region of primary AML cells may lead to impairment of mitochondrial biogenesis (reduction of mtDNA copy number) and derangement of mitochondrial ATP synthesis. During this perturbation, mitochondria in primary AML cells might produce a large amount of reactive oxygen species, which causes the vicious cycle observed in chronic inflammatory diseases and cancers as well. DisclosuresNo relevant conflicts of interest to declare.

Effect of methanol and Me 2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish ( Danio rerio)

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me 2SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP/ATP ratios were assessed following exposure to cryoprotectants for 30 min at room temperature. Results obtained by confocal microscopy showed that 30 min exposure to 2 M methanol induced a loss of membrane potential, although viability tests showed no decrease in survival even after 5 h post-exposure incubation. Higher concentrations of methanol (3 and 4 M) induced not only a decrease in mitochondrial membrane potential but also the loss of mitochondrial distributional arrangement, which suggested a compromised mitochondrial function. Furthermore 3 and 4 M treatments resulted in a decrease in viability assessed by Fluorescein diacetate–Propidium iodide (FDA–PI) and in a decrease in mtDNA copy number and ADP/ATP ratio after 5 h incubation following methanol exposure, indicating a delayed effect. The use of Me 2SO, which is considered to be a more toxic CPA to zebrafish ovarian follicles than methanol, caused a decrease in viability and a sustained decrease in ATP levels accompanied by failure to maintain mtDNA copy number within 1 h post-exposure incubation. These results indicated that even CPAs that are considered to have no toxicity as determined by Trypan blue (TB) and FDA–PI tests can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development.

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts.

In vitro supplementation with dAMP/dGMP leads to partial restoration of mtDNA levels in mitochondrial depletion syndromes

Mitochondrial DNA depletion syndrome, a frequent cause of childhood (hepato)encephalomyopathies, is defined as a reduction of mitochondrial DNA copy number related to nuclear DNA. It was previously shown that mtDNA depletion can be prevented by dAMP/dGMP supplementation in deoxyguanosine kinase-deficient fibroblasts. We investigated myotubes of patients diagnosed with mtDNA depletion carrying pathogenic mutations in DGUOK, POLG1 (Alpers syndrome) and TYMP. Differentiating myotubes of all patients and controls were supplemented with different doses of dAMP/dGMP or dAMP/dGMP/dCMP in TYMP deficiency, and analysed for mtDNA/nDNA ratio and for cytochrome c oxidase (COX) activity. Serum deprivation and myotube formation triggered a decrease in mtDNA copy number in DGUOK or POLG1 deficient myotubes, but not in TYMP deficiency and healthy controls. Supplementation with dAMP/dGMP leads to a significant and reproducible rescue of mtDNA depletion in DGUOK deficiency. POLG1 deficient myotubes also showed a mild, not significant increase in mtDNA copy number. MtDNA depletion did not result in deficient COX staining in DGUOK and POLG1-deficient myotubes. Treatment with ethidium bromide resulted in very severe depletion and absence of COX staining in all cell types, and no recovery was observed after supplementation with dAMP/dGMP. We show that supplementation with dAMP/dGMP increases mtDNA copy number significantly in DGUOK deficient myotubes and, leads to a mild, non-significant improvement of mtDNA depletion in POLG1 deficiency. No adverse effect on mtDNA copy number was observed on high-dose supplementation in vitro. Further studies are needed to determine possible therapeutic implications of dAMP/dGMP supplementation for DGUOK deficiency in vivo.

Paradoxical Effect of Mitochondrial Respiratory Chain Impairment on Insulin Signaling and Glucose Transport in Adipose Cells

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

Role of SUV3 Helicase in Maintaining Mitochondrial Homeostasis in Human Cells

In yeast mitochondria, RNA degradation takes place through the coordinated activities of ySuv3 helicase and yDss1 exoribonuclease (mtEXO), whereas in bacteria, RNA is degraded via RNaseE, RhlB, PNPase, and enolase. Yeast lacking the Suv3 component of the mtEXO form petits and undergo a toxic accumulation of omega intron RNAs. Mammalian mitochondria resemble their prokaryotic origins by harboring a polyadenylation-dependent RNA degradation mechanism, but whether SUV3 participates in regulating RNA turnover in mammalian mitochondria is unclear. We found that lack of hSUV3 in mammalian cells subsequently yielded an accumulation of shortened polyadenylated mtRNA species and impaired mitochondrial protein synthesis. This suggests that SUV3 may serve in part as a component of an RNA degradosome, resembling its yeast ancestor. Reduction in the expression levels of oxidative phosphorylation components correlated with an increase in reactive oxygen species generation, whereas membrane potential and ATP production were decreased. These cumulative defects led to pleiotropic effects in mitochondria such as decreased mtDNA copy number and a shift in mitochondrial morphology from tubular to granular, which eventually manifests in cellular senescence or cell death. Thus, our results suggest that SUV3 is essential for maintaining proper mitochondrial function, likely through a conserved role in mitochondrial RNA regulation.

Physiological and Biochemical Defects in Carboxyl-terminal Mutants of Mitochondrial DNA Helicase

Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general.

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Deficiency of the α Subunit of Succinate–Coenzyme A Ligase Causes Fatal Infantile Lactic Acidosis with Mitochondrial DNA Depletion

Fatal infantile lactic acidosis is a severe metabolic disorder characterized by the onset of lactic acidosis within the 1st d of life and early death. We found a combined respiratory-chain enzyme deficiency associated with mitochondrial DNA (mtDNA) depletion in a small consanguineous family with this disorder. To identify the disease-causing gene, we performed single-nucleotide polymorphism homozygosity mapping and found homozygous regions on four chromosomes. DNA sequencing revealed a homozygous 2-bp deletion in SUCLG1, a gene that encodes the alpha subunit of the Krebs-cycle enzyme succinate-coenzyme A ligase (SUCL). The mtDNA depletion is likely explained by decreased mitochondrial nucleoside diphosphate kinase (NDPK) activity resulting from the inability of NDPK to form a complex with SUCL.

Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis

Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in the brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects.

The 6‐maleimidocaproyl hydrazone derivative of doxorubicin (DOXO‐EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage

Doxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long-term and explain in part the delayed onset of heart dysfunction. DOXO-EMCH a 6-maleimidocaproyl hydrazone derivative of doxorubicin, is an albumin binding prodrug which has entered clinical trials because of its superior antitumor and toxicological profile. In the present work, we examined the chronic cardiotoxicity of DOXO-EMCH in direct comparison with doxorubicin. Rats (11 weeks of age) were treated with intravenous doxorubicin (0.8 mg/kg weekly for 7 weeks), an equimolar dose of DOXO-EMCH (1.1 mg/kg), or with 3.3 mg/kg of DOXO-EMCH. Controls received saline. Animals were euthanized at 48th week. Rats exposed to doxorubicin had a severe clinical, and histopathological cardiomyopathy with depressed myocardial activity of cytochrome c-oxidase (COX, 26% of controls), reduced expression of the mtDNA-encoded COX II subunit, decreased mtDNA copy numbers (46% of controls), and high levels of malondialdehyde and superoxide (787% of controls). All parameters were highly correlated with myocardial damage. Both DOXO-EMCH groups did not differ from controls with regard to clinical symptomatology, mortality and mitochondrial enzymes, although the myocardia of the high-dose group had slightly increased histopathological abnormalities, depressed mtDNA copies (74% of controls) and elevated superoxide levels (347% of controls). Doxorubicin-exposed hearts and to a lesser extent the myocardia of both DOXO-EMCH groups contained mtDNA-deletions. In summary both DOXO-EMCH doses were superior over doxorubicin with respect to clinical and histopathological evidence of cardiomyopathy, myocardial COX-activity, COX II expression, mtDNA-content, mtDNA mutation loads and superoxide production in rats.

Impaired Activity of Mitochondrial Respiratory Chain Enzyme Complexes and Mitochondrial Genomic Aberrations in Leukemia Cells from Patients with Acute Myeloid Leukemia.

Background: Mitochondrial DNA (mtDNA) was particularly susceptible to oxidative damage and mutation because of the high rate of reactive oxygen species (ROS) generation and inefficient mtDNA repair system. Somatic mutations of mtDNA lead to errors in the mtDNA-encoded polypeptide chains belonging to the proton-translocating complexes of the mitochondrial inner membrane. Therefore, we investigated level of ROS, mitochondrial genomic aberrations (mitochondrial genomic instability, mtGI) and the change in enzyme activity of mitochondrial respiratory chain complexes I–IV and in acute myeloid leukemia (AML) cells.Methods: Forty-eight AML bone marrow samples and 57 control blood samples were used after receiving informed consent. The intracellular ROS generation of cells can be investigated using the 2′,7′-dichlorfl uorescein-diacetate and flow cytometry. The results were expressed as mean fluorescence intensity (MFI). We sequenced the mtDNA control region, the tRNA leucine 1 gene plus part of NADH dehydrogenase (ND) 1, and cytochrome b (CYTB) directly and checked the activity of mitochondrial respiratory chain enzyme complexes I to IV using a spectrophotometric kinetic assay. To investigate mtGI, we also examined six mtGI repeats (303 poly C, 16184 poly C, 514CA repeats, 3566 poly C, 12385 poly C and 12418 poly A) using size-based PCR product separation with capillary electrophoresis. mtGI was further confirmed by cloning and sequencing. The mtDNA molecules were analyzed quantitatively using real-time PCR.Results: MFI in AML cells (4,435±709) was significantly higher than those in control blood cells (1,562±141) (P<0.05). Many polymorphisms, as well as new mtDNA variants in the control region and ND1 and CYTB genes were detected in this study. A total of 606 mtDNA sequence variants were identified. Of these, 15 mtDNA variants were identified as novel mutations that were absent from control subjects and established mtDNA polymorphism databases. There were profound alterations of mtGI in the 303 poly C, 16184 poly C, and 514 CA repeats. Seven patients (15%) had leukemia cell-specific mtDNA substitution mutations in the ND1 and CYTB genes. The enzyme activities of AML cells compared with control subjects were 0.828±2.72 vs. −2.48±3.18 for complex I, 0.03±0.08 vs. 0.144±1.72 for complex II/III, and 0.244±0.16 vs. 0.505±0.73 for complex IV. The AML cells showed decreased enzyme activity in respiratory chain complexes I, II, and III. The AML cells had an approximately two-fold decrease in mtDNA copy number compared with the results for control subjects.Conclusion: Mitochondrial genomic aberrations were commonly observed in primary AML cells. High level of ROS in these cells might be an injury to the mitochondrial genome. These mtDNA alterations may impair the activity of mitochondrial respiratory chain enzyme complexes and reduce the mtDNA copy number in patients with AML.

MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion

The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2, SUCLA2, DGUOK and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17. We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice.